Activation Of STAT5 Research Articles

Cell survival signaling in the bovine mammary gland during the transition from lactation to involution

In dairy cows, mammary gland involution, and thus a decline in milk production, occurs following peak lactation. To examine the cell signaling pathways regulating involution of the mammary gland, signal transducer and activator of transcription factors (STAT5 and 3), suppressors of cytokine signaling (SOCS1–3 and CIS), insulin-like growth factors (IGF1 and 2), and protein kinase B (Akt) were examined. Mammary involution was induced by termination of milking, and alveolar tissue was collected from 52 nonpregnant, primiparous, mid-lactation Holstein-Friesian cows killed at 0, 6, 12, 18, 24, 36, 72, and 192h postmilking. Qualitative immunohistochemistry showed that activated (phosphorylated) STAT5-P was localized in nuclei of mammary epithelial cells at the early time points, with detection levels decreasing by 24h postmilking. In contrast, STAT3-P was barely detectable at the early time points, with detection levels increasing following longer postmilking periods. This was supported by Western analysis, which showed a decline in STAT5 and STAT5-P protein levels by 24h postmilking, no change in STAT3 levels, and an increase in STAT3-P protein (barely detectable at the early time points) by 72h postmilking. Quantitative real-time reverse transcription PCR analysis showed SOCS1 and SOCS3 mRNA increased by 72h postmilking compared with 6h postmilking. The SOCS2 mRNA remained unchanged across the time series, whereas CIS decreased by 18h postmilking and remained lower compared with that at 6h postmilking until 72h postmilking. The IGF1 mRNA increased by 192h postmilking, whereas IGF2 mRNA decreased by 18h postmilking compared with 6h postmilking. The IGFBP5 mRNA and protein levels of Akt and Akt-P remained unchanged over the time series. These results show that reciprocal activation of STAT5 and STAT3 occurs at the onset of mammary gland involution in the bovine, albeit at a slower rate than in rodents. Mathematical modeling of the pathways indicated that activated STAT3 could block the STAT5 pathway by upregulating SOCS3. The regulation of IGF1-Akt signaling suggests that by 192h postmilking in dairy cows, the involution process is still in the reversible phase, with quiescent mammary epithelial cells not yet in the senescent phase.

IL-10-Induced miR-155 Targets SOCS1 To Enhance IgE-Mediated Mast Cell Function.

IL-10 is an important regulatory cytokine that modulates a wide range of immune cells. Whereas it is best known for its ability to suppress immune responses, IL-10 has been found to be pathogenic in several human and animal studies of immune-mediated diseases. There is a considerable gap in our understanding of the molecular mechanisms behind the stimulatory effects of IL-10 during allergic inflammation. IL-10 treatment has been shown to suppress mast cell TNF production. In this study, we report that whereas TNF secretion was reduced, IL-10 surprisingly enhanced IgE-mediated protease and cytokine production both in vitro and in vivo. This stimulatory effect was consistent in mouse and human skin mast cells. IL-10 enhanced activation of the key FcεRI signaling proteins Stat5, JNK, and ERK. We demonstrate that IL-10 effects are dependent on Stat3 activation, eliciting miR-155 expression, with a resulting loss of suppressor of cytokine signaling-1. The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155-deficient mice. Taken together, our results reveal an IL-10-induced, Stat3-miR-155 signaling pathway that can promote mast cell responses.

Luminal STAT5 mediates H2AX promoter activity in distinct population of basal mammary epithelial cells.

Deregulated STAT5 activity in the mammary gland causes parity-dependent tumorigenesis. Epithelial cell cultures transfected with constitutively active STAT5 express higher levels of the histone H2AX than their non-transfected counterparts. Higher H2AX expression may be involved in tumorigenesis. Here, we aimed to link high STAT5 activity to H2AX–GFP expression by looking for distinct types of mammary cells that express these proteins. In vitro and in transgenic mice, only 0.2 and 0.02%, respectively, of the cells expressed the H2AX–GFP hybrid gene. Its expression correlated with that of the endogenous H2AX gene, suggesting that detectable H2AX–GFP expression marks high levels of H2AX transcript. Methylation of the H2AX promoter characterized non-GFP-expressing H2AX–GFP cells and was inversely correlated with promoter activity. Administration of 5-azacytidine increased H2AX promoter activity in an activated STAT5-dependent manner. In transgenic mice, H2AX–GFP expression peaked at pregnancy. The number of H2AX–GFP-expressing cells and GFP expression decreased in a Stat5a-null background and increased in mice expressing the hyperactivated STAT5. Importantly, H2AX–GFP activity was allocated to basal mammary cells lacking stem-cell properties, whereas STAT5 hyperactivity was detected in the adjacent luminal cells. Knockdown of RANKL by siRNA suggested its involvement in signaling between the two layers. These results suggest paracrine activation of H2AX via promoter demethylation in specific populations of basal mammary cells that is induced by a signal from neighboring luminal cells with hyper STAT5 activity. This pathway provides an alternative route for the luminally confined STAT5 to affect basal mammary cell activity.

Deciphering the Different Types of Refractory Celiac Disease

Universite Paris Descartes-Sorbonne Paris Centre Gastroenterology department, Hopital Europeen Georges Pompidou APHP, Paris Inserm U989, Hopital Europeen Georges Pompidou APHP Paris, France Fifteen years ago has emerged the concept of refractory celiac disease (RCD). [1,2] RCD is defined by persisting malabsorption and villous atrophy after one year of strict GFD ascertained by a dietician. RCD has been subdivided into two subgroups according to the normal (type I RCD (RCDI)) or abnormal phenotype of intraepithelial lymphocytes (IEL) (type II RCD (RCDII). RCDI is indistinguishable from active CD and is characterized by a polyclonal repertoire. Survival of patients with RCDI is slightly inferior to that of those with celiac disease. [3,4] In contrast, RCDII is a severe enteropathy with frequent intractable ulcerative duodeno-jejunitis. RCDII is characterized by a clonal repertoire and is considered as a low-grade intraepithelial lymphoma. [1,2] The five year survival rate of patients with RCDII is around 50%. This poor prognosis is due to severe malnutrition and increased risk of overt lymphoma. One of the current challenges in RCD is to ascertain the type of RCD. Indeed, diagnosis of RCD has become highly specialized. It requires small bowel endoscopic specialized investigations such as capsule endoscopy and double balloon enteroscopy. [5,6] Intestinal biopsy needs to be studied by different complementary techniques such as immunohistochemistry, flow cytometry, and Multiplex PCR, all necessary to ascertain the precise diagnosis. Recent studies have reported case series of chronic intestinal diseases mimicking the both types of RCD. RCDI needs to be distinguished from other causes of non clonal villous atrophy such as enteropathy associated with common variable immunodeficiency (CVID). [7] Demonstration of HLA haplotypes encoding HLA-DQ2 or DQ8 can be useful as their absence excludes CD or RCD as a cause of villous atrophy. [8] Nevertheless, genotypes HLA-DQ2/DQ8 are also found in 77% of CVID patients with enteropathy mimicking CD defined by intestinal intra-epithelial lymphocytosis. [7] So evidence of serum hypogammaglobulinemia and histopathological features such as intestinal plasmocytic rarefaction or nodular lymphoid hyperplasia appear essential to distinct CVID enteropathy from RCDI. [7] RCDII needs to be distinguished from other cases of clonal enteropathy with villous atrophy. Flow cytometry allows to tell the difference between RCDII and other intestinal clonal malignancies with villous atrophy. Indeed, demonstration of excess of CD4+IEL with a specific Vbeta repertoire is essential for diagnosis of intestinal CD4 lymphoproliferations. [9] Diagnosis may be particularly tricky when CD4 lymphoproliferation [9] or large granular lymphocytic leukemia (LGL) [10] complicate an authentic CD. Consequently, spectrum of RCD has been recently substantially extended. Advances in accuracy of diagnostic tools could reduce the number of undiagnosed RCD and increase the frequency of RCD currently estimated to 1 to 2% of CD patients. [11,12] Besides epidemiological interest, improvement of diagnostic tools allowed precise diagnosis of RCD type and appropriate treatment. By example, distinction between RCDII and CD complicated by LGL is crucial as cyclosporin, inefficient in RCDII, is in contrast very useful for treating LGL. Indeed, treatment of RCD remains another challenge particularly in case of RCDII patients who have poor prognosis and for whom referral treatment does not yet exist. Immunosuppressors are poorly efficient and may possibly trigger overt lymphoma. [13] Purine analogues such pentostatine or cladribine (2 CDA) have been largely used in the past with slight therapeutic effect. [4,14,15] Autologous haematopoietic stem cells transplantation represents an interesting alternative but when combined to chemotherapy for hopping sustained reduction of abnormal IEL. [16,17] We are currently evaluating this strategy in a prospective phase II trial. Many advances in the understanding of the pathogenesis of RCDII were made by deciphering the anti-apoptotic signaling pathway of the cytokine IL-15 which prevents the elimination of IEL in CD and RCDII. [18] The IL-15 induced antiapoptotic signaling pathway includes activation of IL15Rβγ, Jak3, STAT5 and Bcl-xL [19] which represent so many therapeutic targets. Blocking the effect of IL-15 appears of a particular interest in RCDII but may probably be useful in other types of RCD with possible involvement of IL-15 such as RCDI or LGL-complicated CD. [20] Continuing the characterization of the different forms of RCD will probably guide next pathogenic studies and permit to increase our therapeutic efficacy in the future.

Enhanced IFN-γ signals and STAT5-phosphatase dependent attenuated IL-2 signals in the pathogenesis of rheumatoid arthritis.

Abstract The importance of type I interferons as a driver of pathogenesis in autoimmunity, including rheumatoid arthritis (RA) is well recognized. However, in this context the contribution of type II interferons (IFN-γ) has been equivocal. This study developed from our recent discovery that the expression of IFN-γ receptor gene Ifngr1 strongly associates with RA disease and expression of Ifngr2 associates with radiographic severity. In this study we show that IFN-γ induced activation is significantly greater in naïve, central memory, Tfh and Treg subsets of CD4+ T cell populations from RA patients compared to healthy controls (HC). Remarkably, such enhanced IFN-γ induced STAT1 activation was not observed in CD8 T cell populations from RA patients. The activation of STAT1 and consequently, IFN-γ signals are regulated by tyrosine phosphatases. Treatment with phenyl-arsine oxide (PAO), a broadly active phosphatase inhibitor, had no effect on IFN-γ induced STAT1 activation in any T cell population from RA or HC. In contrast, such treatment with PAO greatly enhanced IL-2 induced activation of STAT5 in RA CD4 T cell populations (naïve, central memory effector memory, Treg, Tfh) but not from HC or in CD8 T cell populations. This result suggests that the regulatory activity of IL-2 in RA is dampened by a STAT5-specific phosphatase. We further determined that such a STAT5-phosphatase was also expressed and active in regulatory and effector CD4 T cell populations from the spontaneously autoimmune BXD2 mice. Overall our results indicate that CD4 T cell subpopulation dependent enhanced IFN-γ signals and dampened IL2-STAT5 signals contributes to pathogenesis in RA.

Overexpression of total STAT1 during lymphopenia-induced proliferation leads to decrease CD4 T cell survival

Abstract To preserve the size and diversity of the T cell repertoire throughout life, homeostatic mechanisms regulate both survival and proliferation through IL-7 and TCR signaling. IL-7 signals through a heterodimer receptor composed of the IL-7Rα and the γc chains. Binding of IL-7 to its receptor leads to activation of STAT5 and induction of target genes implicated in survival and proliferation signals. IL-7/IL-7R signaling can also activate STAT1 and STAT3 but the function of IL-7-dependent activation of these transcription factors is largely unknown. Under lymphopenic conditions, IL-7 modulates the expression levels of total STAT1 (t-STAT1) leading to an IL-7-dependent STAT1 signaling in addition to STAT5. We generated a transgenic mouse that overexpress STAT1 in CD4 and CD8 T cells to understand the contribution of IL-7-dependent STAT1 activation on T cell homeostasis in the setting of lymphopenia. We found that under lymphopenia-induced proliferation T cells from the STAT1 transgenic mouse upregulates t-STAT1 leading to diminish CD4 T cell survival. This mechanism could be a physiological mechanism to regulate the expansion of the CD4 T cell pool under lymphopenic conditions.

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs. CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812. In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib. CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.

Designing an In-Silico Mimetic for Thrombopoietin Using Combinatorial Library

Differentiation and proliferation of megakaryocytic progenitor cells into mature platelets is triggered and activated upon binding of Thrombopoietin Receptor (TPOR), a cytokine receptor protein, to thrombopoietin (TPO), a 95 KD glycoprotein hormone.TPO plays a very important role as it can easily direct Hematopoietic Progenitor Cells into the megakaryocytic lineage but even then very few mimetic have been reported, Agonists such as Romiplostim and Electrombopag are reported, recombinant TPO mimetic are also available but structural mimetic is only TMP, so the scope of further research is available. A promising approach for designing a mimetic can be targeting the binding site of the receptor such that the mimetic peptide has similar agonist activity as the ligand. Diverse libraries can be created using in-silico approaches and they are analyzed to bind receptor efficiently. In this study efforts are made for the development of TPO mimetic with therapeutic potential. TPO increases its number, prevent apoptosis, and thereby increases platelet production. Activation of TPO receptor leads to phosphorylation of the cytoplasmic domain as well as downstream activation of JAK2, STAT5, and other anti-apoptotic pathways. This increases the viability of stem cells and precursors of all lineages. The anti-apoptotic effect of TPO is probably important in patients with ITP: bone marrow megakaryocytes and megakaryocyte precursors are already increased but these cells are undergoing apoptosis mediated by anti-platelet IgG and T cells, thereby limiting platelet production. Administration of TPO decreases the apoptotic rate of the megakaryocyte precursors and thereby enhance the platelet production. Once produced, platelets are still affected by TPO. TPO’s new Combinatorial library was designed by interaction sites of TPOR with TPO and previously reported TPO mimetic peptide (TMP), further screening of combinatorial librarywas done and an efficient mimetic was chosen based on scoring function commonly called Energy. Energy is calculated in terms of KJ/mol, based on the orientations generated by docking ligand with the receptor, the most efficient ligand has the most stable conformation with minimum energy value. In this study docking of TPO and TPOR has a minimum energy value of -518.20; TPOR and mimetic designed has a total score of -456.55which is better than the known mimetic TMP, which has an energy score of -170.17 with TPOR. Computational approaches confirmed that the resulting peptide mimetic was stable and similar in nature to TPO. Further Ex-vivo study of designed mimetic can confirm its role in regenerative medicine, increasing the shelf life of platelet to improve cellular viability of platelet by preventing programmed cell death, i.e. apoptosis, which can be achieved by adding TPO during storage since TPO levels increase when platelet count is low, to stimulate additional production. TPO is absorbed by transfused platelets, which may decrease available TPO levelor in the disease condition.

Interfacial geometry dictates cancer cell tumorigenicity

Within the heterogeneous architecture of tumour tissue there exists an elusive population of stem-like cells that are implicated in both recurrence and metastasis. Here, by using engineered extracellular matrices, we show that geometric features at the perimeter of tumour tissue will prime a population of cells with a stem-cell-like phenotype. These cells show characteristics of cancer stem cells in vitro, as well as enhanced tumorigenicity in murine models of primary tumour growth and pulmonary metastases. We also show that interfacial geometry modulates cell shape, adhesion through integrin α5β1, MAPK and STAT activity, and initiation of pluripotency signalling. Our results for several human cancer cell lines suggest that interfacial geometry triggers a general mechanism for the regulation of cancer-cell state. Similar to how a growing tumour can co-opt normal soluble signalling pathways, our findings demonstrate how cancer can also exploit geometry to orchestrate oncogenesis.

BM microenvironmental protection of CML cells from imatinib through Stat5/NF-κB signaling and reversal by Wogonin.

Constitutive Stat5 activation enhanced cell survival and resistance to imatinib (IM) in chronic myelogenous leukemia (CML) cells. However, the mechanism of Stat5 activation in mediating resistance to IM in bone marrow (BM) microenvironment has not been evaluated precisely. In this study, we reported HS-5-derived conditioned medium (CM) significantly enhanced IM resistance in K562 and KU812. Interestingly, upregulation of the proportion of CD34+ subpopulation was found in CML cells. Subsequently, the BCR/ABL-independent activation of Stat5 increased P-glycoprotein (P-gp) activity in CM-mediated protection of CML stem cells (LSCs) from IM. Further research revealed Stat5 activation increased the DNA binding activity of NF-κB though binding of p-Stat5 and p-RelA in nucleus. Moreover, highly acetylated RelA was required for Stat5-mediated RelA nuclear binding. The study further confirmed that Wogonin potentiated the inhibitory effects of IM on leukemia development by suppressing Stat5 pathway both in CM model and the K562 xenograft model. In summary, results clearly demonstrated BCR/ABL-independent Stat5 survival pathway could contribute to resistance of CML LSCs to IM in BM microenvironment and suggested that natural durgs effectively inhibiting Stat5 may be an attractive approach to overcome resistance to BCR/ABL kinase inhibitors.

IL-8 induces miR-424-5p expression and modulates SOCS2/STAT5 signaling pathway in oral squamous cell carcinoma

Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR-424-5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR-424-5p. The miR-424-5p-induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR-424-5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR-424-5p expression could be induced by the cytokine IL-8 primarily through enhancing STAT5 transcriptional activity rather than NF-κB signaling. Antagomir-mediated inactivation of miR-424-5p prevented the IL-8-induced cell migration and invasion, indicating that miR-424-5p is required for IL-8-induced cellular invasiveness. Taken together, these data indicate that STAT5-dependent expression of miR-424-5p plays an important role in mediating IL-8/STAT5/SOCS2 feedback loop, and scavenging miR-424-5p function using antagomir may have therapeutic potential for the treatment of OSCC.

Apontic regulates somatic stem cell numbers in Drosophila testes.

BackgroundMicroenvironments called niches maintain resident stem cell populations by balancing self-renewal with differentiation, but the genetic regulation of this process is unclear. The niche of the Drosophila testis is well-characterized and genetically tractable, making it ideal for investigating the molecular regulation of stem cell biology. The JAK/STAT pathway, activated by signals from a niche component called the hub, maintains both germline and somatic stem cells.ResultsThis study investigated the molecular regulation of the JAK/STAT pathway in the stem cells of the Drosophila testis. We determined that the transcriptional regulator Apontic (Apt) acts in the somatic (cyst) stem cells (CySCs) to balance differentiation and maintenance. We found Apt functions as a negative feedback inhibitor of STAT activity, which enables cyst cell maturation. Simultaneous loss of the STAT regulators apt and Socs36E, or the Stat92E-targeting microRNA miR-279, expanded the somatic stem cell-like population.ConclusionsGenetic analysis revealed that a conserved genetic regulatory network limits JAK/STAT activity in the somatic stem cells of Drosophila testis. In these cells, we determined JAK/STAT signaling promotes apt expression. Then, Apt functions through Socs36E and miR-279 to attenuate pathway activation, which is required for timely CySC differentiation. We propose that Apt acts as a core component of a STAT-regulatory circuit to prevent stem cell overpopulation and allow stem cell maturation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0103-3) contains supplementary material, which is available to authorized users.

Prolactin inhibits a major tumor-suppressive function of wild type BRCA1

Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.

Abstract IA20: Genomics of a STAT1 knockout mouse model of human ER+ breast cancer

Abstract Estrogen receptor alpha positive luminal breast cancers are the most frequent subtype of breast cancer. Previous work has established that Stat1-/- mouse mammary tumor model recapitulates signaling, expression and phenotypic alterations observed in this subtype of human breast cancers. To identify transforming events that contribute to tumorigenesis, we performed whole genome sequencing of 22 Stat1-/- primary mammary tumors and cell lines. We discovered a novel hotspot of somatic mutations in 100% of tumors that resulted in a truncated form of the prolactin receptor (Prlr). Targeted sequence analysis identified similar mutations in 77.8% of ductal carcinoma in situ. Co-expression of truncated and full-length Prlr in normal cells led to activation of oncogenic Stat3 and Stat5 as well as cellular transformation. In conclusion, truncating mutations of Prlr drive tumor development in a model of human ERa+ breast cancer and should be considered as novel antitumor targets. Citation Format: Elaine Mardis, Obi L. Griffith, Ruby Chan Szeman, Malachi Griffith, Kilannin Krysiak, Zachary Skidmore, Jasreet Hundal, Julie A. Allen, Arthur Cora, Alexander P. Miceli, Heather Schmidt, Lee Trani, Krishna-Latha Kanchi, Christopher A. Miller, David E. Larson, Robert S. Fulton, Richard K. Wilson, Robert D. Schreiber. Genomics of a STAT1 knockout mouse model of human ER+ breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA20.

IL-7 signalling represses Bcl-6 and the TFH gene program

The transcriptional repressor Bcl-6 is linked to the development of both CD4+ T follicular helper (TFH) and central memory T (TCM) cells. Here, we demonstrate that in response to decreased IL-2 signalling, T helper 1 (TH1) cells upregulate Bcl-6 and co-initiate TFH- and TCM-like gene programs, including expression of the cytokine receptors IL-6Rα and IL-7R. Exposure of this potentially bi-potent cell population to IL-6 favours the TFH gene program, whereas IL-7 signalling represses TFH-associated genes including Bcl6 and Cxcr5, but not the TCM-related genes Klf2 and Sell. Mechanistically, IL-7-dependent activation of STAT5 contributes to Bcl-6 repression. Importantly, antigen-specific IL-6Rα+IL-7R+ CD4+ T cells emerge from the effector population at late time points post influenza infection. These data support a novel role for IL-7 in the repression of the TFH gene program and evoke a divergent regulatory mechanism by which post-effector TH1 cells may contribute to long-term cell-mediated and humoral immunity.

Preclinical assessment of clinic ready compounds for the treatment of Spinal Muscular Atrophy

Event Abstract Back to Event Preclinical assessment of clinic ready compounds for the treatment of Spinal Muscular Atrophy Faraz T. Farooq1, 2* 1 Emirates College for Advanced Education, Science and Mathematics Education, United Arab Emirates 2 Children's Hospital of Eastern Ontario (CHEO), Research Institute, Canada Background: Spinal muscle atrophy (SMA) is an autosomal recessive neurodegenerative disease which is characterized by the loss of α motor neurons resulting in progressive muscle atrophy (1). The loss of functional Survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene is the cause of SMA (2). Method: A potential treatment strategy for SMA is to upregulate levels of the SMN protein originating from the copy gene SMN2 which could compensate in part for the absence of the functional SMN1 gene (3-5). Goal of the study and Results: We have shown a novel therapeutic strategy for SMA treatment through the activation of the p38 and STAT5 kinase pathway activating clinic ready compounds (6, 7). Celecoxib (p38 activating drug) and Prolactin (STAT5 activator) demonstrate a clear promise for use in SMA clinical trial studies as they increase SMN protein levels, ameliorate disease phenotype in SMA mouse model, have blood brain barrier penetration and also have FDA approved status (6, 8). We have also identified other clinic ready STAT5 and p38 pathway activators as new candidates for SMA treatment (9, 10). These results provide evidence and need for FDA approved compound library screen to identify clinic ready compounds for the treatment of SMA. Conclusion: SMA is a deadly neurodegenerative disease with a prevalence of 1:10000 mostly affecting children and more than 50% affected children die before the age of two (11-16). It is the most common genetic cause of infant death and is currently untreatable (15). This study will help in the identification of clinic ready compounds as new candidates for SMA treatment which will provide us an excellent platform to extend this approach on a wider scale to other rare disorders. We believe it shall bring effective treatments closer to reality for rare genetic disorders. References

How Intrinsic Molecular Dynamics Control Intramolecular Communication in Signal Transducers and Activators of Transcription Factor STAT5.

Signal Transducer and Activator of Transcription STAT5 is a key mediator of cell proliferation, differentiation and survival. While STAT5 activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated with a broad range of hematological and solid tumor cancers. Therefore the development of compounds able to modulate pathogenic activation of this protein is a very challenging endeavor. A crucial step of drug design is the understanding of the protein conformational features and the definition of putative binding site(s) for such modulators. Currently, there is no structural data available for human STAT5 and our study is the first footprint towards the description of structure and dynamics of this protein. We investigated structural and dynamical features of the two STAT5 isoforms, STAT5a and STAT5b, taken into account their phosphorylation status. The study was based on the exploration of molecular dynamics simulations by different analytical methods. Despite the overall folding similarity of STAT5 proteins, the MD conformations display specific structural and dynamical features for each protein, indicating first, sequence-encoded structural properties and second, phosphorylation-induced effects which contribute to local and long-distance structural rearrangements interpreted as allosteric event. Further examination of the dynamical coupling between distant sites provides evidence for alternative profiles of the communication pathways inside and between the STAT5 domains. These results add a new insight to the understanding of the crucial role of intrinsic molecular dynamics in mediating intramolecular signaling in STAT5. Two pockets, localized in close proximity to the phosphotyrosine-binding site and adjacent to the channel for communication pathways across STAT5, may constitute valid targets to develop inhibitors able to modulate the function-related communication properties of this signaling protein.

Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization.

Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability.

FLT3-ITD Confers Resistance to Bortezomib By Protecting the mTOR/4EBP1 Pathway through Activation of STAT5 and Induction of Pim-1 Expression

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in AML, with the former strongly associated with a poor prognosis. We have recently revealed that FLT3-ITD confers resistance to the PI3K/AKT pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in AML. The proteasomal inhibitor bortezomib (BZM) has recently been reported as a promising agent for treatment of AML. We examine here the molecular mechanisms involved in induction of apoptosis by BZM in hematopoietic cells, including AML cells, and evaluate the effects of FLT3-ITD and TKD on these mechanisms and on the sensitivity of cells to BZM-induced apoptosis.We first comparatively examined the effect of BZM on survival of hematopoietic 32D cells and human leukemic UT7 cells driven by FLT3-ITD (32D/ITD and UT7/ITD) or FLT3-D835Y (32D/TKD and UT7/TKD). BZM induced activation of Bax, decline in mitochondrial membrane potential, and activation of caspase-9, thus leading to apoptosis, more conspicuously in cells driven by FLT3-ITD than FLT3-TKD. These results indicate that FLT3-ITD, as compared with FLT3-TKD, conferred resistance to apoptosis induced by BZM through the intrinsic pathway in these cells.To elucidate the molecular mechanisms involved in BZM-induced apoptosis in these cells, we next examined the effect of BZM on expression levels of FLT3 in these cells as well as in human leukemic MV4-11 cells expressing FLT3-ITD. Intriguingly, treatment of these cells with BZM conspicuously reduced expression levels of FLT3. It was further reveled that BZM further facilitated the decline in FLT3-ITD expression in MV4-11 cells treated with actinomycin D to shut down the transcriptional activity. Furthermore, BZM significantly retarded the recovery of FLT3-ITD expression in MV4-11 cells washed out from the translation inhibitor cycloheximide. These results suggest that BZM may downregulate the FLT3-ITD expression mainly at the translational level. However, we did not observe any significant difference in extent of the BZM-induced decline in expression levels between FLT3-ITD and FLT3-TKD.We next examined the effect of BZM on the mTOR/4EBP1 pathway, which we have shown to play important roles in regulation of apoptosis downstream of FLT3-ITD. It was found that BZM downregulated this pathway more significantly in 32D/TKD cells than in 32D/ITD cells. Because we have also previously found that STAT5 activated robustly by FLT3-ITD plays an important role in modulation of the mTOR/4EBP1 pathway, we examined the effects of BZM on 32D/TKD cells forced to express the constitutively activated STAT5 mutant, STAT5A1*6. As expected, STAT5A1*6 conferred resistance to BZM-induced downregulation of the mTOR/4EBP1 pathway as well as apoptosis in 32D/TKD cells. Consistent with this, the STAT5 inhibitor pimozide, clinically in use for neuropsychiatric disorders, abrogated the resistance of 32D/ITD, UT7/ITD, and MV4-11 cells to BZM-induced inhibition of the mTOR/4EBP1 pathway and apoptosis.We finally examined the possible involvement of the STAT5 target gene product Pim-1 in acquisition of resistance to BZM by cells expressing FLT3-ITD. We fist confirmed that Pim-1 was expressed at a higher level in 32D/ITD cells than in 32D/TKD cells and that STAT5A1*6 increased the expression level of Pim-1 in 32D/TKD cells. We then examined the effects of a specific Pim kinase inhibitor, AZD-1208, and found that it synergistically downregulated the mTOR/4EBP1 pathway and induced apoptosis with BZM in 32D/ITD cells as well as 32D/TKD cells expressing STAT5A1*6. We also examined the effects of a BET bromodomain inhibitor, JQ1, which has recently been shown to inhibit the STAT5 activity and to reduce specifically the expression level of Pim-1 as well as c-Myc in MV4-11 cells. As expected, pretreatment of 32D/ITD or MV4-11 cells with JQ1 conspicuously sensitized these cells to BZM-induced apoptosis.These results suggest that BZM downregulates FLT3 expression and the mTOR/4EBP1 pathway to activate the intrinsic apoptotic pathway and that robust STAT5 activation by FLT3-ITD confers resistance to BZM on AML cells through protection of the mTOR/4EBP1 pathway at least partly by inducing Pim-1 expression. The present study may contribute to development of novel therapeutic strategies against FLT3-ITD-positive AML by combined use of BZM and the STAT5/Pim-1 pathway inhibitors. DisclosuresNo relevant conflicts of interest to declare.

CITED2 Cooperates with Low PU.1 and DNMT3A to Maintain Self-Renewal in Hematopoietic Stem Cells

CITED2 has a conserved role in the maintenance of normal hematopoiesis. We have recently shown that ~70% of acute myeloid leukemia (AML) patients display enhanced CITED2 expression levels. Interfering with CITED2 expression is detrimental for leukemia maintenance in vitro and in vivo, demonstrating that CITED2 is critically important for the survival of leukemic stem cells (LSCs). Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) led to significantly better human engraftment in transplanted NSG mice, consistent with the maintenance of very primitive lin- CD34+ CD38- CD90+ CD45RA- HSCs within the bone marrow 28 weeks after transplantation. Although the CITED2-engrafted mice displayed enlarged spleens, blood development appeared normal, as measured through myeloid, B and T cell staining. This indicates that CITED2 as a single hit is not sufficient to transform human CD34+ cells.CITED2 expression frequently coincides with low expression of the myeloid transcription factor PU.1, suggesting that combined effects, rather than single events are important during AML development. To investigate this, we combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and studied hematopoietic development. CITED2 increased the percentage of immature CD34+ CD38- cells 5-fold, which was not further increased by the additional downregulation of PU.1. However, functional analysis through limiting dilution LTC-iC assays indicated that combining PU.1 down-, with CITED2 upregulation led to a synergistic 8.5-fold increase in LTC-iC frequency, whereas only changing PU.1 or CITED2 induced a respective 1.4 to 3-fold change in HSC frequency. To more stringently assess self-renewal, we cultured transduced cells for 4 weeks on MS5 cells under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently performed CFC assays. Whereas after 4 weeks all groups displayed similar colony numbers, secondary and tertiary replatings demonstrated that self-renewal could only be maintained for more than 10 weeks when CITED2 upregulation was combined with PU.1 downregulation. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+ CD38- HSCs, as replating of CD34+ CD38+ progenitor-derived colonies did not yield new CFCs.In order to investigate the underlying mechanisms, we performed transcriptome analysis on human HSCPs after knockdown of PU.1, overexpression of CITED2 or the combination of both. PU.1Low-CITED2High cells displayed a gene expression pattern different from the PU.1Low or CITED2High only cells, suggesting that the two events have synergistic effects. Some genes, like HLX and SF3B1 have been shown to cause or are mutated in AML, demonstrating that the synergistic changes are related to AML. When comparing the differentially regulated genes in the PU.1Low -CITED2High cells to the gene expression in the Hemaexplorer database, a similar pattern was observed, when compared between AML and normal cells.In order to investigate the effects of the PU.1low CITED2high combination on AML development, we resorted to a PU.1-dependent mouse model of AML development. CITED2 expression in BM cells from PU.1KD/KD mice (in which deletion of an Upstream Regulatory Element leads to an 80% downregulation of PU.1), led to a steady increase of GFP+ cells over time as compared to control cells and demonstrated a dramatic expansion of Gr-1+ Mac-1+ cells, a hallmark of AML in these mice. This suggests that CITED2 contributes to a faster progression towards AML upon lowering of PU.1.To identify if our model corresponds to AMLs with a specific subset of mutations, we clustered publically available AML data (TCGA), based on the gene expression changes in the PU.1Low -CITED2High cells. The majority of AMLs clustered together in 2 groups, in which FLT3, p53 and DNMT3A mutations were most prevalent. FLT3 mutations, through its activation of STAT5, are consistent with high CITED2 expression, whereas p53 mutations are consistent with our data indicating that CITED2 loss regulates HSCs in a p53-dependent manner. The presence of DNMT3A mutations suggests that DNA methylation changes collaborate with high CITED2 and low PU.1 during leukemogenesis. This is currently under investigation.In summary, our data imply that CITED2, low PU.1 and potentially changes in DNA methylation all contribute to maintenance of self-renewal and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.