- Jak-Stat Signalling
- Overall Concepts
Jak-Stat Signalling: from Basics to Disease
The particular strength of the proposed research is to apply the fundamental knowledge about Jak-Stat mechanisms in cellular processes to the next level of complexity. The general objective of the proposed SFB is to jointly investigate how Jaks and Stats regulate immunity to infection, inflammation and cancer. Our unifying aim is to both study the topics individually and the links between them. Our concepts are supported by emerging similarities between features of acute inflammation and cancer progression, and the association of signal transduction that originate form infection and inflammation with tumourigenesis. We will analyse contributions of Jaks and Stats to cell autonomous mechanisms of tumourigenesis and distinguish these from contributions to cancer immuno-surveillance or the establishment of an inflammatory environment promoting cancer growth. Our studies will consider a role for hitherto poorly understood interactions with Jak-Stat partner molecules and they will test potential functions of non-tyrosine-phosphorylated Stats and kinase-inactive Jaks. Furthermore, they will address mechanisms by which Stats regulate their target gene expression.
Our specific objectives are:
- Linking Jak or Stat modifications to target gene activity and biological functions
- Defining Jak and Stat functions in shaping cell and organ reactivity in disease
- Unravelling the Janus-faced character of Jaks and Stats in diseases: preventors and drivers of disease
Topics of research area ‚infection and immunity’:
- Lymphocyte Stat1 and immunity against Listeria monocytogenes
- Gene regulation by Stat1 isoforms in immunity against infection
- Kinase-inactive Tyk2 and IFN-II production-mediated immunity
Topics of research area ‚hematopoietic malignancies’:
- Oncogenic Jak2 and/or Stat5: cooperation with haplo-insufficient loss of tumour suppressors
- Tumour suppressing activity of Stat1 in leukemogenesis
- NK cell-mediated tumour surveillance: canonical and non-canonical Tyk2 or Stat1 activities
Topics of research area ‚non-canonical Jak-Stat activity’:
- Stat1 isoform-specific transcriptional activity
- Stat1 / NFκB cooperativity and chromatin remodelling
- Tyk2 and Stat1: non-canonical effects on NK cell biology
The SFB-JakStat centers research competence in the field of cytokine and growth factor signaling covering immunity to infection, inflammation and cancer up to novel non-canonical Jak-Stat actions causing disease. SFB-JakStat formed a network of nine research groups, which either had already a profound molecular background in Jak-Stat signaling or which were coming from other research areas newly focusing their expertise on Jak-Stat and contributing to the success of the consortium as is evident by joint publications. SFB-JakStat served also as a communication platform for Jak-Stat research and many international experts were invited to Vienna to share and discuss their projects. This enabled the five participating institutions (University of Vienna, Medical University of Vienna, University of Veterinary Medicine Vienna, Institute for Molecular Pathology and Ludwig Boltzmann Institute for Cancer Research) to achieve high quality research in close collaborations, to exchange expertise and to generate new knowledge in the following areas: (i) Jak-Stat in inflammation and immune responses; (ii) Jak-Stat in hematopoiesis and leukemogenesis; (iii) Jak-Stat in carcinoma and (iv) Jak-Stat in novel mechanisms of action. In particular, Tyk2 and Stat1 were studied with respect to antimicrobial innate and adaptive immune responses, T cell-mediated host defense and defense against tumors by T- and NK cells. The role of Jak2, Stat1, Stat3 and Stat5 was further defined for leukemogenesis and normal hematopoiesis. Stat3 and Stat5 signaling was investigated in the context of inflammation-induced liver damage and cancers associated with inflammation, wound repair, fibrosis and metastasis. New interferon target genes - most notably the anti-inflammatory gene tristetraprolin - were found as a consequence of Stat1 transcriptional synergies. Mouse models to study Tyk2 kinase-dependent and -independent functions were generated and are ready to be studied in inflammation, cancer and infectious diseases. In addition, the combined efforts in the SFB-JakStat mouse core facility resulted in the availability of several more Stat1 and Tyk2 mutants of high relevance for future projects. The SFB-internal Scientific Advisory Board and the FWF-organized reviewing process with international renowned reviewers evaluated the outcome and future perspectives of the SFB-JakStat projects and was impressed by the overall performance of the consortium. Albeit being a basic research and mouse models orientated program, the research track record of the participants and the research environment provided by the host institutions will ensure the translation of SFB-JakStat results into clinical or pharmaceutical relevance.
Our specific objectives were combined to the work packages (WPs):
- JakStat in inflammation and immune respones focusing on innate and adaptive immunity against microbes and tumours
- JakStat in hematopoiesis and leukemogenesis
- JakStat in carcinoma focusing on liver cancer and mechanisms of epithelial-mesenchymal transition (EMT)
- JakStat in novel mechanisms of action focusing on structure/function interrelations, interaction partners, phosphorylation or enzymatic requirements, target genes and mechanisms of target gene induction.
Organization of the SFB
The SFB-JakStat is hosted by the Vetmeduni Vienna and the Speaker is Mathias Müller (MM). The administrative officer Regina Kadi is located on the Vetmeduni campus and is responsible for communicating with the secretaries and the offices for financial and personnel affairs of the legal entities of the research groups and with the funding organization, the FWF. Scientific communication and dissemination is largely performed through the internet platform www.jak-stat.at.
The group leaders of the project parts form the Executive Board, which is headed by the Speaker in close consultation with the co-speaker, Thomas Decker (TD). The Executive Board elaborates and approves scientific, personnel and financial affairs and consults with the international Scientific Advisory Board (SAB).
The SAB is composed of Christian Bogdan (Mikrobiologisches Institut, Universitätsklinikum Erlangen, DE), Meinrad Busslinger (Research Institute of Molecular Pathology (IMP) Vienna, AT), Tony Green (Cambridge Institute for Medical Research, University of Cambridge, UK), Florian Greten (II. Medizinische Klinik und Poliklinik (Gastroenterologie), Technische Universität München, DE), Klaus Pfeffer (Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Heinrich-Heine-Universität Düsseldorf, DE) and Valeria Poli (Molecular Biotechnology Center and Dept. of Genetics, Biology and Biochemistry, University of Turin, I). Towards the end of the 2nd funding period a SAB meeting was organized along the lines of the review hearings for SFBs held by the Austrian Science Fund, FWF. The SAB’s comments on the success of the previous funding period and the potential of the projects proposed for the final funding period form the basis for the present application.
Scientifically, the work of the SFB-JakStat is divided into three Research Areas: (i) Jak-Stat in Infection and Immunity; (ii) Jak-Stat in Cancer and Immune Surveillance; and (iii) Non-canonical Mechanisms of Jak-Stat Action. In the second funding period the consortium consisted of four groups headed by Thomas Decker (TD, Max F. Perutz Laboratories, MFPL, University of Vienna), Richard Moriggl (RM, Ludwig Boltzmann Institute of Cancer Research (LBI-CR) Ludwig Boltzmann Society), Veronika Sexl (VS, Institute of Pharmacology and Toxicology, Vetmeduni Vienna) and Mathias Müller (MM, Institute of Animal Breeding and Genetics, Vetmeduni Vienna) and his Co-PI Birgit Strobl (BS, Institute of Animal Breeding and Genetics, Vetmeduni Vienna). In accordance with the recommendations of the reviewers of the previous application, reflected in the decision of the FWF Board, the projects of the junior group leaders Robert Eferl (RE, LBI-CR) and Wolfgang Mikulits (WM, Institute of Cancer Research, Medical University of Vienna) were assigned to the coordination project and were undertaken under the guidance of the Speaker and Deputy Speaker.
Short- and long-term scientific perspective
The year 2011 marked the 20th anniversary of the Jak-Stat signal tranduction pathway. The pathway was originally identified in mammalian cells but it rapidly became evident that Jaks and Stats are conserved in organisms ranging from slime molds (harbouring Stats only) and insects to vertebrates. In all of these organisms, most complex biological processes from embryonic development to ageing are influenced by Jak and/or Stat signaling. The Viennese Jak-Stat consortium is pooling intellectual and technological efforts to understand how Jaks and Stats regulate immunity to cancer, inflammation and infection and act variously as oncogenes or as tumor suppressors. The consortium members share a longstanding track record in Jak-Stat signaling and complement and mutually stimulate one another as they stem from disciplines as apparently diverse as medical pharmacology and oncology (VS), infection biology (BS), microbiology, immunity and gene regulation (TD), tumor biology (RM) and biotechnology and transgenetics (MM). Each of the SFB-JakStat members greatly benefits from the combined interdisciplinary knowledge and the complementary technological skills available and accessible within the consortium.
The strength of the research programme was and remains the strictly Jak-Stat-centric view on disease causalities and resolutions. Classical Jak-Stat functions include the cytokine receptor-associated catalytic activity of Jaks and the activation of Stats through phosphorylation on tyrosine and serine residues. Recently, it has become clear that Jaks-Stats also have a number of non-canonical activities, which we define as kinase- and/or receptor-independent functions of Jaks or phosphorylation-independent functions of Stats. The SFB-JakStat has substantially contributed to this emerging field through comprehensive in vivo studies on U- (unphosphorylated) Stat1 and kinase-inactive Tyk2 in tumor and infection settings. The research has been enabled by gene-modified mice, which have been specifically generated by the consortium. In addition, the SFB-JakStat has gained an internationally high reputation through its published work on Stat1 and Tyk2 in bacterial infections and tumor surveillance, on Stat3 and Stat5 in hepatic and intestinal cancers and inflammations and on Stat5 in leukemogenesis. These previous achievements and the extensive collection of mouse mutants form the basis for the SFB-JakStat’s short-term scientific perspective, which is to unravel the molecular interactors, networks, targets and chromatin dynamics of non-canonical Stat1 and Tyk2 functions and to extend the knowledge of the molecular functions of Jaks and Stats as drivers or suppressors in cancers and infections. In addition the international visibility of the SFB-JakStat enables the consortium members to pursue research projects based on initial findings from the SFB-JakStat as “stand-alone” or collaborative research projects in association to the SFB, thereby further strengthening the network. The long-term perspective is to apply the fundamental knowledge of non-/canonical Jak-Stat mechanisms and their cross-talk to other signaling cascades in cellular und systemic processes to identify novel targets for pharmacological interference with deregulated Jaks and/or Stats that are associated with pathological conditions in man.
Scientific development of the SFB
In the second funding period the SFB-JakStat was organized into the three Research Areas (i) Infection and Immunity, (ii) Cancer and Immune Surveillance, and (iii) Non-canonical Mechanisms of Jak-Stat Action. The infection and immunity research (i) strongly focused on Stat1 and Tyk2 and was contributed to by project parts F2803 (TD) and F2808 (MM, BS). Both groups used the intracellular bacterium Listeria monocytogenes to work on the cell type-specific interdependence of Stat1 and interferon (IFN) type I production and response during infection. The unexpected finding of an IFN-I-independent role of Stat1 in lymphocyte-mediated immunity to L. monocytogenes will be pursued in the forthcoming funding period. Analysis of Tyk2 during bacterial and viral infections revealed complex requirements for a protective innate immune response and a homeostatic metabolic response during infection. The surprisingly clear contribution of kinase-inactive Tyk2 to the development of septic shock will be further investigated in a stand-alone project to be associated with the SFB. The research on Jak-Stat in carcinogenesis and cancer-associated inflammation and immunity (ii) focused on Stat3 and Stat5 in hepatic and gastrointestinal cancers, which was investigated by the project parts F2801 (RE, WM) and F2807 (RM); on Jak2 and Stat5 in hematopoietic malignancies, studied by project parts F2807 (RM) and F2810 (VS); on Stat1 as a tumor suppressor in breast cancer, which was the focus of work by VS, TD and F2808 (MM, BS); and on Stat1 and Tyk2 as essential players in NK cell-mediated tumor surveillance, an investigation to VS, BS, TD and MM contributed. Complex studies were published showing the interplay of Stat5 with growth hormone and glucocorticoid responses in the development of liver cancer and demonstrating that Stat3 can have a tumor-suppressing or -promoting function in liver and intestinal cancer, depending on the precise nature of the carcinogenic stimulus. The work on breast, liver and intestinal cancer will be continued in projects to be undertaken in association with the SFB but funded from other sources. The functions of Jaks and Stats in NK cells will be further studied in the next funding period. The research on non-canonical Jak-Stat functions (iii) concentrated on U-Stat1 and kinase-inactive Tyk2 in NK cell activation and on (un-)phosphorylated Stat1 and Stat1 isoforms in promoter cooperativeness. Both areas were collaborative efforts of TD, VS, BS and MM. With respect to U-Stat1 and kinase-inactive Tyk2 we detected an unexpected phosphorylation-independent function in tumor immunosurveillance for both molecules and this surprising finding will form the focus of work in the forthcoming funding period. With regard to Stat1 and promoter activation by interaction with other transcription factors we have published two widely acclaimed papers. We established furthermore that the current notion that Stat1α is an active and Stat1β an inhibitory form in the IFN-II response has to be revisited, as Stat1β knock-in mice show IFN-II-induced transcription profiles that translate in vivo into substantial antibacterial defense. These topics continue to be of major interest in the next funding period.
Short summary of the project parts
SFB-P01MM ‘Coordination Project’ ensured the efficient operation and the smooth scientific progress of the SFB-JakStat. During the funding period there were no scientific or financial anomalies that required corrective measures from SFB-P01 or the Executive Board. SFB-P01 organized and coordinated the regular monthly SFB-JakStat seminars, which featured progress reports from SFB members as well as presentations from invited speakers, and the regular meetings of project leaders. A retreat of the principal investigators with an extensive SWOT analysis and a detailed evaluation by the Scientific Advisory Board were instrumental in controlling the quality of the SFB’s performance and in defining its future scientific strategy.
Project Part 01 – Stat3 Functions in Inflammation-induced Liver- and Intestinal Cancer (Robert Eferl & Mathias Müller)
The focus of the research is the role of Stat3 in hepatoprotection, liver cancer and intestinal cancer. Mice with conditional inactivation of Stat3 in hepatocytes and cholangiocytes (Stat3Δhc) were generated using the AlfpCre transgene and were crossed to Mdr2 knockout (Mdr2-/-) mice to investigate the function of Stat3 in hepatoprotection and inflammation-induced liver cancer. Loss of Stat3 in hepatocytes and cholangiocytes of Mdr2-/- mice led to premature lethality associated with strongly aggravated hepatic fibrosis. Moreover, a conditional mouse model was established to study the function of Stat3 in intestinal cancer. Conditional inactivation of Stat3 in intestinal epithelial cells reduced formation of early adenomas in ApcMin mice but promoted progression and invasiveness of advanced cancers.
Project Part 01 – The Pro- and Anti-Oncogenic Role of Stat3 in Liver Cancer Progression (Wolfgang Mikulits & Mathias Müller)
We have addressed the oncogenic role of Stat3 in Ras-mediated HCC progression. We found that constitutively active (ca)Stat3 is tumor-suppressive in Ras-transformed p19ARF-/- hepatocytes, while expression of Stat3 lacking Tyr705 phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3Δhc/p19ARF-/- hepatocytes (lacking Stat3 and p19ARF) exhibit increased tumor growth compared to those expressing Stat3, demonstrating a tumor suppressor activity of Stat3 in cells lacking p19ARF. p14ARF (the human homologue of p19ARF) was knocked down in Hep3B hepatoma cells associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Using kinase inhibitors we showed that Jaks cause pY-Stat3 activation independently of p14ARF levels, indicating that p14ARF controls the oncogenic function of pY-Stat3 downstream of Jaks. The data provide evidence that ARF determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression. Further examinations will focus on the identification of pY-Stat3- and U-Stat3-depending target genes collaborating with ARF and oncogenic Ras in liver cancer and will be pursued in a stand-alone project in association with the SFB.
SFB-P03TD ‘Regulation of Genes and Immunity by Canonical and Non-Canonical Stat1 Signaling’ contributed to Research Areas (i) and (iii) and aimed to investigate the many facets of Stat1 activity in the immune system. To this end we study the response of mice with various Stat1 mutations or deficiencies to infection with the intracellular bacterial pathogen Listeria monocytogenes. Using mice carrying a conditional Stat1 deletion we determined the requirement of Stat1 signaling in innate responses to L. monocytogenes. Intriguingly however, Stat1 deletion in T cells led to an increase of resistance to L. monocytogenes.
Mechanisms providing RNA polymerase II (Pol II) with the competence to clear promoters of infection-induced Stat1 target genes and to create the competence for transcript elongation were compared between genes induced by the ISGF3 (Stat1/Stat2/IRF9) complex alone or through functional cooperativity between ISGF3 and NFκB. We demonstrated that the need for Stat/NFκB interaction arises from shared activities in the assembly of a functional transcription pre-initiation complex (PIC), where ISGF3 stimulates TFIID and Pol II binding only after NFκB recruited the kinases necessary for the phosphorylation of the Pol II carboxyterminal domain. Finally, we revealed a role for transcription factor IRF7 and its interaction with the Stat1 dimer in macrophage activation by IFNγ and a hitherto undescribed role for tonic kinase signaling in the regulation of IRF7 activity during the response to IFNγ.
SFB-P07RM ‘Leukemogenesis Studies with Persistently Activated Forms of Stat5 and the GH-Jak2-Stat5 Interplay in Liver Cancer Development’ largely contributed to the Research Area (ii). Results from our work in the liver brought new evidence for the oncogenic role of hyper growth hormone (GH) signaling and a novel role of Stat5 and glucocorticoid receptor (GR) proteins as tumor suppressors during liver carcinogenesis. Moreover, our results shed light on the functions of GH and corticosteroid signaling for metabolism, hepatoprotection, inflammatory conditions and post-natal body growth control. Concerning hematopoietic cancer we found that serine phosphorylation of Stat5a is crucial for myeloid transformation. Together with SFB-P10VS we showed that increased Stat5 expression levels are associated with tyrosine kinase inhibitor resistance and that the Bcr/Abl translocation product is strictly dependent on Stat5. Oncogenic Stat5 which resides in the cytoplasm as a tyrosine phosphorylated molecule in many myeloid neoplasms was found capable to activate PI3K-AKT-mTOR and combined inhibition of mTOR signaling and survival protein function was found to block myeloid transformation. Moreover, we established new mouse models for persistent Stat5 signaling and crossed an inducible Stat5 mouse model to PTEN tumor suppressor mice, which resulted in elevated white blood cells. The transgenic mouse models of oncogenic Stat5a action will be continued in the future.
SFB-P08MMBS ‘Fine-tuning the Reverse Genetics of Tyk2 and Stat1’ contributed to all three Research Areas. We published the initial characterisation of a genetically engineered mouse model expressing the kinase-inactive form of Tyk2. Tyk2K923E mice provide a highly valuable tool to dissect kinase-dependent (canonical) and kinase-independent (non-canonical) functions of Tyk2 in vivo and to study the side effects and efficacy of the Jak inhibitors under development. With respect to IFNα/β signaling kinase-inactive Tyk2 is not able to compensate for the loss of Tyk2. However, kinase-inactive Tyk2 could control tumor growth to a similar extent as wild-type Tyk2 and, partially contributes to the pathogenesis of sepsis. This clearly confirms our hypothesis that inhibition of Tyk2 kinase activity does not mimic Tyk2 deficiency. Concerning Stat1 we used isoform-specific knock-in mice (Stat1Δα/Δα and Stat1Δβ/Δβ) and challenged the current belief that Stat1β acts as dominant negative isoform if activated as homodimers, i.e. after IFNγ treatment, by demonstrating that Stat1β alone mounts an IFNγ-mediated antibacterial response in vivo. These exciting findings in Tyk2 and Stat1 signaling prompted us to divide our future work in research proposals concentrating on either Tyk2 (MM) or Stat1 (BS).
SFB-P10VS ‘Jak-Stat Involvement in Leukemogenesis and NK-mediated Tumor Surveillance’ contributed to Research Area (ii). We identified Stat5 as crucial signaling node downstream of the Bcr/Abl-induced oncogene that drives the onset and the maintenance of disease. Thus we define Stat5 as potential therapeutic target. We further noticed that the Bcr/Abl tyrosine kinase itself phosphorylates Stat5, and only in the initial transformation phase the Stat5 canonical up-stream kinase Jak2 is required. The key role for Stat5 in Bcr/Abl induced disease is underlined by the fact that tyrosine kinase inhibitor resistance can be caused by high Stat5 expression. Leukemic cells are recognized and eliminated by NK cells. To study NK cell-mediated tumor surveillance we generated Ncr1-Cre mice that express Cre recombinase exclusively in NK cells. This allowed us to define the absolute requirement for Stat5 within the NK cell compartment. Mice deficient for Stat5 in NK cells lack mature NK cells and are incapable to fight NK cell-surveilled tumors. In addition we studied the role of Stat1 in NK cells. Here we found that Cdk8-dependent phosphorylation of a critical serine site in the transactivation domain of Stat1 (S727) inhibits NK cell activity. Accordingly, mice carrying a point mutation in this serine site (Stat1-S727A) are largely protected to develop NK cell-surveilled tumors.
Organization of the SFB
The hosting institution of the SFB-JakStat continues to be the Vetmeduni Vienna, which employs the speaker Mathias Müller (MM). The administrative officer Regina Kadi communicates with the secretariats and the offices for financial and personnel affairs of the research groups and the funding organization. Scientific communication and dissemination is largely performed through the internet platform www.jak-stat.at.
The remaining leaders of the project parts belong to Vetmeduni Vienna (Veronika Sexl VS, Birgit Strobl BS), Max F. Perutz Laboratories (MFPL) University of Vienna (Thomas Decker TD), Center for Molecular Medicine (CeMM) Austrian Academy of Science (Robert Kralovics RK) and Ludwig Boltzmann Institute for Cancer Research (LBI-CR) (Richard Moriggl RM). Together they form the Executive Board, which is headed by the speaker in close consultation with the co-speaker TD. The Executive Board elaborates and approves the scientific, personnel and financial affairs and consults with the Scientific Advisory Board (SAB). The SAB continues to consist of Christian Bogdan (Erlangen, DE), Meinrad Busslinger (Vienna, AT), Tony Green (Cambridge, UK), Florian Greten (Munich, DE), Klaus Pfeffer (Düsseldorf, DE) and Valeria Poli (Turin, I).
Short- and long-term goals of the SFB research programme
The research programme briefly outlined above will be organized in three Research Areas: (i) Infection and Immunity, (ii) Hematopoietic Malignancies and Tumor Immune Surveillance and (iii) Molecular Mechanisms of Canonical and Non-canonical Jak-Stat Action. The Viennese Jak-Stat consortium combines intellectual and technological efforts to understand how Jaks and Stats regulate immunity to cancer, inflammation and infection and act as oncogenes or tumor suppressors. The SFB-JakStat members greatly benefit from the range of interdisciplinary knowledge and the complementary technological skills within the consortium.
A new asset is the connection to human genetics provided by SFB-P12RK. This subproject aims to examine causal relationships between Jak mutations and secondary chromosomal deletions occurring as hematopoietic tumors progress towards a state of severe malignancy. This task will be made possible by the collective expertise of SFB-JakStat and a unique collection of genetically modified mice.
The short-term scientific perspectives are driven by a common aim to develop our research areas, focussing on the most intriguing findings from the previous funding period. In research area (i) the following main hypotheses will be tested:
- Lymphocyte Stat1 drives a gene expression programme that weakens the contribution of this cell type to immunity against Listeria monocytogenes (beyond the detrimental activity noted for type I IFN).
- Target genes of the Stat1β isoform contribute to immunity against infection.
- Kinase-inactive Tyk2 contributes to immunity against infection through a partial rescue of IFNγ production.
In research area (ii) the following main hypotheses will be tested:
- Stat5 activity, and the oncogenic potential of the Jak2-Stat5 axis, are increased by haplo-insufficiency of tumor suppressor genes and a corresponding loss of tumor suppressor proteins.
- Stat1 is a suppressor of leukemogenesis.
- NK cell-mediated tumor immune surveillance is regulated by canonical and non-canonical Stat1 and Tyk2 signaling.
Research area (iii) will test the following main hypotheses:
- Despite the lack of its C-terminal TAD, Stat1&beta contains a transcriptional transactivation function that may differ from that of Stat1α.
- The ISGF3 complex (containing Stat1) recruits RNA polymerase II through the Mediator complex. A class of genes requires ISGF3 and NFκB to interact in the assembly of clearance and elongation-competent promoter chromatin.
- U-Stat1 contributes to gene expression required for the development of NK cell cytotoxicity.
The national and international visibility of the SFB-JakStat enables the consortium members to pursue research projects based on initial findings inside of SFB-JakStat as stand-alone or collaborative research projects associated with the SFB.
The long-term perspective remains to apply fundamental knowledge of non-/canonical Jak-Stat mechanisms and their cross-talk to other signaling cascades and to cellular und systemic processes. Our findings will be of value for the identification of novel pharmacological targets and for drug-mediated interference with pathophysiology caused by deregulated Jaks and/or Stats.
An important stronghold of the SFB remains the common Technology Platform. Importantly, the impressive collection of mice with Jak-Stat deficiencies or mutations has been further expanded during the second funding period. An important aspect of the Technology Platform will be the ability to provide in-house bioinformatics support that will be applied to results obtained by next generation sequencing.
In summary, the SFB will continue to bundle the resources and expertise of six groups in four different institutions for mutual benefit, added value and increased international competitiveness and to achieve a better prospect of translating Jak-Stat research into clinical application.
Brief summary of the individual project parts and contribution of the individual project parts to the common goal
F2801_MM (Mathias Müller) and the administration officer (a) will organize the monthly scientific and administrative SFB meetings by all members and co-workers and the invitation of external speakers on a regular basis. The monthly meeting of the project leaders, co-applicants and co-workers ensures continuous monitoring of the overall quality and progress of the project parts. In addition the coordinator organizes a Scientific Advisory Board meeting to evaluate the programme according to the FWF guidelines at least once during the funding period. SFB-P01MM (b) heads and co-ordinates the Technology Platform consisting of ‘Gene-modified Mice/Biomodels Austria’, ‘Bioinformatics’ and ‘Advanced Tissue Culture’. SFB-P01MM (c) acts as a central communication platform linking partner institutions, the scientific community and local, national, international or government institutions, third party organizations, industries or media. SFB-P01MM (d) will promote dissemination of results to the scientific community and the general public mainly through www.jak-stat.at and (e) will support the career management of young scientists.
F2803_TD (Thomas Decker) ‘Regulation of genes and immunity by canonical and non-canonical Stat1 signaling’ is embedded in Research Area (i) and (iii). Stat1 deficiency in humans or mice decreases innate resistance against intracellular microorganisms. The hypotheses underlying the first part of project SFB-P03TD are based on our previous finding that deletion of Stat1 in T lymphocytes enhances clearance of the intracellular bacterium Listeria monocytogenes and survival of infected mice. A major aim of our proposal is therefore to understand how lymphocyte Stat1 weakens immunity to intracellular bacteria. The second part of the proposal addresses mechanistic aspects of the antimicrobial transcriptional response. Stat1 regulates antimicrobial genes in L.monocytogenes-infected macrophages, both in its dimeric form and as a subunit of transcription factor ISGF3. We will examine the molecular mechanisms allowing ISGF3 to recruit RNA polymerase II (Pol II) into the transcription pre-initiation complex (PIC). Because a significant number of antimicrobial genes require Stat1 to interact with transcription factor NFκB we will investigate individual and cooperative participation of ISGF3 and NFκB in promoter activation Pol II competence for transcriptional clearance and elongation. Our studies will contribute insights into molecule and cell-based regulation of the antimicrobial response and thus to the infection and immunity research area of SFB-JakStat.
F2807_RM (Richard Moriggl) ‘Leukemogenesis studies with persistently activated forms of Stat5 (and the GH-Jak2-Stat5-GR interplay in liver cancer development)’ is central for Research Area (ii). Hematopoietic cancers display specific genetic defects and aberrant response to growth factors and cytokines. Clonal variants can transform to leukemia or lymphoma. The Jak2-Stat5 pathway drives several core cancer pathways, but it can also promote differentiation or senescence. The main hypothesis is that Jak2-Stat5 signaling is only oncogenic in context with loss of tumor suppressor protein function, which represents the majority of mutations found in cancer. Moreover, we hypothesize that Stat5 activity is gradually increasing in the clonal population upon hematopoietic cancer progression. We developed transgenic mice with hematopoietic expression and activation of oncogenic Stat5 to test mono-allelic loss of important tumor suppressor proteins (IKZF1, CUX1, PTEN) or the oncogenic Jak2V617F kinase. We expect cooperativity with gain of function mutations in the Stat5 pathway to study induced core cancer pathways in neoplastic cells.
F2808_MM ‘Fine-tuning the reverse genetics of Tyk2’ contributes to all three Research Areas with the focus on the Jak family member Tyrosine Kinase 2 (Tyk2). The specific goals are (i) the determination of Tyk2 kinase-independent functions in NK cells and their contribution to tumor immune surveillance, and (ii) the dissection of Tyk2 kinase-dependent and -independent functions in the immune defence against L. monocytogenes. The experiments are based on the use of mice expressing a kinase-inactive version of Tyk2 (Tyk2K923E) and on conditional Tyk2 knockout mice (Tyk2fl/fl). Key methodologies include primary cell culture techniques, expression profiling, cytotoxicity assays, FACS, immuno-histochemistry and -pathology, in vivo infections and tumor inductions/transplants (leukemia/lymphoma).
F2810_VS (Veronika Sexl) ‘Jak-Stat involvement in leukemogenesis and NK-mediated tumor surveillance’ contributes to Research Area (ii) and (iii) and is involved in all projects concerning NK cell biology, tumor surveillance and leukemogenesis. In her project she will investigate the role of canonical and non-canonical Stat1 for myeloid leukemogenesis, as Stat1-deficient mice spontaneously succumb to myeloproliferative diseases (MPD) developing into leukemia when aging. She will test the hypothesis that Stat1 is an important tumor suppressor and aim at identifying the cellular compartment and molecular mechanisms how Stat1 fulfils this task. In addition she will investigate the role of canonical and non-canonical Stat1 in NK cell mediated tumor surveillance. Key methodologies include murine leukemia models, in vivo transplantation studies, primary cell culture, FACS and SORT analysis, immunohistochemistry, ChIP-Seq analysis.
F2811_BS (Birgit Strobl) ‘The role of Stat1 isoforms in immunity to infections and in cancer’ is embedded in all three Research Areas and investigates the biology of the alternative splicing products Stat1α and Stat1β, which are identical except for the lack of 38 amino acids at the C-terminus of Stat1β. The key hypotheses tested are (i) Stat1 isoforms transcriptional activation capacity is determined by the promoter context, (ii) Stat1β alone contributes to the host immune defence during infectious diseases, and (iii) Stat1α and Stat1β contribute independently to carcinogenesis and/or tumor immune surveillance. Primary cells of Stat1Δβ/Δβ and Stat1Δα/Δα knockin mice will be analyzed by extensive expression profiling and ChIP technologies. Isoform-specific functions will be monitored through viral (Vesicular Stomatitis Virus, Murine Cytomegalovirus) and bacterial (Listeria monocytogenes) in vivo challenge models followed by immuno-pathology and -histochemistry. Carcinogen-induced tumorigenesis and tumor transplants will be used in the knockin mice.
F2812_RK (Robert Kralovics) ‘Collaboration of Jak-Stat pathway mutations and gene deletions in leukemia’ is central for Research Area (ii). Myeloproliferative neoplasms (MPN) are characterized by increased production of clonal myeloid cells and frequent transformation to acute myeloid leukemia. An oncogenic mutation of Jak2 (Jak2V617F) is frequently involved in these diseases promoting polycythemia vera, essential thrombocytemia or primary myelofibrosis. In addition, hemizygous deletions or mutations of tumour suppressor genes (TSGs) such as IKZF1, CUX1, FOXP1, ETV6, JARID2 and RUNX1 were often found associated with Jak2V617F in patients. The combined genetic mutation status is implicated to aggravate disease to leukemic transformation. We test this mutational interplay by genetic models based on established transgenic mice that allow to question if haplo-insufficient loss of tumorsuppressors on top of the Jak2V617F driver mutation is the cause for aggressive cancer formation. We combine Jak2V617F knockin mice with haplo-insufficient loss of tumor suppressor genes, namely IKZF1, CUX1, FOXP1 and ETV6 to validate clinical observations found in patient with hematopoietic neoplasms. We further postulate that haplo-insufficient loss of these tumor suppressor genes will increase activation and signaling through the Jak-Stat5 axis, best measurable by RNA-seq expression profiling.
For a more detailed report including references see ‚NEWS’ section.
General concept and overall results
Absence or malfunction of JAK-STAT pathways causes many diseases ranging from immunological deficiencies, deregulated inflammatory responses, auto-immunity, anaemia, hypo- or hyperplasia, malignant cell transformation and metabolic disorders. In particular, we focused on the JAK-STAT family members JAK2, TYK2, STAT1, STAT2, STAT3, STAT5A and STAT5B in three major Research Areas: (i) JAK-STAT in Infection and Immunity; (ii) JAK-STAT in Cancer (Hematopoietic Malignancies; Gastrointestinal Cancers) and Tumour Immune Surveillance; (iii) the Molecular Mechanisms of Canonical and Non-canonical JAK-STAT Actions.
(i) Jak-Stat in Infection and Immunity (Inflammation)
The initial focus of this area was in the impact of Stat1/Tyk2 mediated IFN signalling and IFN response effectors on intracellular bacteria, in particular Listeria spp., on viral infections and systemic inflammations. The expertise of TD, BS, MM, PK were combined and unravelled tissue specific actions of Stat1 and Tyk2 and IFN response molecules (Costantino et al., 2008; Gratz et al., 2011; Kernbauer et al., 2012; Kratochvill et al., 2011; Radwan et al., 2010; Schaljo et al., 2009; Stockinger et al., 2009; Zwaferink et al., 2008). This SFB focus was expanded to the more general aspect of the interaction between the immune system and gut microbiota. The consequences of disturbing Jak-Stat signalling for the composition of the gut microbiota and for the host responses in acute and chronic colitis were interrogated. Our work defined the importance of Jak-Stat signals upon all types of IFNs in for the course and outcome of colitis (Rauch et al., 2015). The concerted activities of SFB enabled us to transit to more a global view on microbial induced chromatin and transcriptome changes (see below Research Area (iii)).
(ii) Jak-Stat in GI Cancer Hematopoietic Malignancies and Tumour Immune Surveillance
In this area of research the SFB succeeded in making major contributions within the area of hematopoietic malignancies and tumour development within the gastro-intestinal (GI) tract. In a teamed effort the labs of RM and VS studied the role of JAK2/STAT5 in hematopoietic tumours. STAT5 was defined as potential therapeutic target and critical modifications of STAT5 were evaluated for their potential to serve as novel therapeutic points of attack (Hoelbl et al., 2010; Warsch et al., 2012; Warsch et al., 2011). Based on the ground breaking work of the RM lab that uncovered that serine phosphorylation in STAT5 is required for STAT5 dependent leukemogenesis (Friedbichler et al., 2010), the VS lab subsequently defined PAK1 kinases as major druggable upstream regulators (Berger et al., 2014a). Moreover RM and VS contributed to clarify the role of JAK2 in Bcr/Abl induced disease, which has been a long standing matter of debate. In fact JAK2 is not required in Bcr/ABl induced disease maintenance, but deletion of JAK2 in in vivo models turned out detrimental as it accelerated disease (Grundschober et al., 2014; Hantschel et al., 2012; Warsch et al., 2013). These findings are of great impact for the design of clinical studies implying JAK inhibitors that are currently ongoing. VS is considered a pioneer in the field of NK cell dependent tumour surveillance supported by TD, BS and MM. VS, TD, BS and MM succeeded in unravelling the role of JAK-STAT signalling in NK cell biology over the last years. Whereas STAT5 is a master player in NK cell development and survival, STAT3 suppresses NK cell cytotoxicity (Eckelhart et al., 2011; Gotthardt et al., 2014). VS, TD and MM also managed to identify CDK8 dependent serine phosphorylation of STAT1 as important regulator of NK cell mediated tumour surveillance (Putz et al., 2013). This observation attracted significant interest from the pharmaceutical industry and several companies are now in contact to exploit CDK8/STAT1S727 as novel therapeutic axis to support the immune system in its fight against cancer. The enormous importance of the immune system for cancer control is recently more and more recognized. Lastly, the lab of VS defined a non- canonical and kinase-independent function for the cell cycle kinase CDK6 as transcriptional regulator. CDK6 has common transcriptional targets with STAT3 to promote hematopoietic tumour formation (Kollmann et al., 2013). This finding and the expertise on JAK2-STAT5 signalling opened a novel area of research that allowed VS, RM and RK to participate in the SFB-MPN and VS to apply for an ERC-advanced grant.
In the field of GI cancers we focused on chronic injury conditions (fibrosis and colits) that promote hepatocellular carcinoma (HCC) or colorectal cancers (CRC). Genetically modified model systems based on induced and spontaneous carcinogenesis revealed for the first time the essential tumour suppressive function of STAT3 in HCC upon LOF of ARF or in CRC upon GOF of WNT signalling in ApcMin mice (Fischer et al., 2007; Mair et al., 2010; Musteanu et al., 2010; Musteanu et al., 2012; Pathria et al., 2015; Schneller et al., 2011). In addition, we unravelled LOF of STAT5 consequences with or without cooperativity of the glucocorticoid receptor in inflammatory HCC upon hyper growth hormone signalling (Friedbichler et al., 2012; Mueller et al., 2011; Mueller et al., 2012).
(iii) Molecular Mechanisms of Canonical and Non-canonical Jak-Stat Actions
Genuine SFB achievements stem from analyses of mouse strains expressing STAT1 phospho-site mutants or isoforms and kinase-inactive TYK2. The work significantly enlightened the consequences of STAT phosphorylation for chromatin changes and transcriptional programs. Furthermore it established a transcriptional activity of the STAT1β isoform in vivo formerly reported to be inactive or even repressive (Bancerek et al., 2013; Ramsauer et al., 2007; Sadzak et al., 2008; Semper et al., 2014). Finally, the promoter specific cooperativity between ISGF3 and NFκB on a mechanistic level to integrate independent signals, allowing for a microbe adapted cellular response, was defined (Farlik et al., 2010; Wienerroither et al., 2014; Wienerroither et al., 2015). For TYK2 we reported the first in vivo non-canonical function in NK cell mediated tumour restriction. Strikingly, kinase-inactive TYK2 restored cytotoxicity without correcting the signalling defects of LOF of TYK2 (Prchal-Murphy et al., 2015).
Definitive erythropoiesis depends on EPOR and JAK2 function, but the unique role of STAT5 was questioned by compensatory STAT3 activation. We showed that GOF of STAT5 restored EPOR or JAK2 deficiency during definitive erythropoiesis (Grebien et al., 2008). Furthermore, we showed an essential function o STAT5 for iron metabolism regulating both the transferrin receptor and iron regulatory proteins (Kerenyi et al., 2008). During the last funding phase we discovered in international collaborations that STAT gene dosage controls inflammation or intestinal epithelial stem cell functions in homeostasis which in particular impacts during injury (Gilbert et al., 2015; Gilbert et al., 2012; Nivarthi et al., 2015).
The strength of SFB-JakStat was and remains the pathway-centric view on molecular mechanistics, disease causalities and therapeutic interventions. The national and international renowned collaborative network that developed over the past decade will be further fostered by the long-term members of the consortium. The regular meetings with external speakers and progress reports of the JAK-STAT centric groups will be perpetuated. Most importantly, the core members of the consortium have sumitted a follow-up SFB concept termed Monarchie and Hierarchies in Shaping Chromatin Landscapes (see NEWS section).